1. Field of the Invention
The present invention relates to a method for the treatment of a sample containing biomolecules. The invention further relates to a method for the lysis of a biological sample, a method for the stabilisation of nucleic acids and/or proteins, a method for the reduction of inhibiting effects in a sample containing nucleic acids and/or proteins, a method for the differential masking of nucleic acids and analysis methods which build on the previous methods or incorporate them.
2. Description of Related Art
It has been known for a long time that the genetic origin and the functional activity of a cell can be determined and examined by studies of biomolecules as e.g. its nucleic acids or proteins. The analyses of these molecules enable the direct access to the origin of the activities of cells. They are thereby potentially superior to indirect conventional methods as e.g. the detection of metabolites. This has led to a wide distribution of nucleic acid and protein analyses in previous years. The biomolecular analyses are thus already used in many areas, e.g. in medical and clinical diagnostics, in pharmaceutics in the development and evaluation of drugs, in food analysis and during the supervision of food production, in agriculture during the breeding of agricultural crop and farm animals and in the environment analysis and in many research areas.
The activities of genes can for example be determined directly by the analysis of the RNA, in particular the mRNA in cells. The quantitative analysis of transcript samples (mRNA samples) in cells by modern molecular-biological methods as e.g. real-time reverse transcriptase PCR (“Real-Time RT-PCR”) or gene expression chip analyses enables e.g. the recognition of wrongly expressed genes whereby e.g. metabolic disorders, infections or the formation of cancer can be recognised. The analysis of the DNA, e.g. from cells, by molecular biological methods as e.g. PCR, RFLP, AFLP or sequencing enables e.g. the proof of genetic defects or the determination of the HLA-type and other genetic markers.
The analysis of genomic DNA and RNA is also used for the direct detection of infectious agents, such as viruses, bacteria etc.
It is the condition for all analysis methods of biomolecules from biological samples that these biomolecules as e.g. DNA, RNA or proteins are made accessible for the corresponding analysis method. Contents of cells or organisms can usually only be analysed when the contents are present in the analysis medium, that is, are e.g. transferred from the cell or the organism into the analysis medium. The cells/organisms are disintegrated for this purpose, so that the contents are not present within but outside of the organism or the cells and the contents of the organism or the cell are freely accessible for the analysis. The disintegration of organisms or parts of organisms (e.g. cells) is also called lysis, the disintegrated organisms are also called lysates.
The disintegration of samples, e.g. organisms, should take place as completely as possible on two counts: (1) All areas of the sample, e.g. of the organism should be disintegrated, so that the contents of possibly all parts of an organism are released in the sample. (2) The disintegration of the sample, e.g. of the organisms, is only completed when the contents were made accessible for the analysis. While the organisms were disintegrated, but the contents still covered, e.g. by cell compartments, an analysis of the contents cannot be carried out in a quantitative manner.
Presently, several lysis methods are known. A cleaning of the contents to be analysed is followed in the most lysis methods, so as to free the contents from all materials which counteract an analysis. Some examples are to be described in the following:
Lysis with detergents: Detergents are amphipatic molecules which dissolve the hydrophobic membrane of cells so that the contents of the cells outpour into the environment. E.g., non-ionic and ionic detergents belong to these. The detergents triton-X100, Nonidet-P40, sodiumdodecyl sulfate (sodiumdodecyl sulfate, SDS), or also cationic detergents as e.g. N-cetyl-N,N,N-trimethyl-ammonium bromide (CTAB) amongst others have further use for the disintegration of organisms. Many methods with cationic detergents were described and patented for the lysis of organisms and for the isolation of nucleic acids (NA). Nucleic acids can e.g. be complexed by cationic detergents for their protection and cleaning. The methods described in the following US patents and US patent applications are part of these methods: U.S. Pat. No. 6,602,718, U.S. Pat. No. 6,617,170, U.S. Pat. No. 5,010,183, U.S. Pat. No. 5,985,572, U.S. Pat. No. 5,300,635, U.S. Pat. No. 5,728,822, and US 2002/0146677 A1, US 2004/0048384 A1, US 2004/0115689 A1, US 2004/0014703 A1. The lysis method with ammonium salts as e.g. N-cetyl-N,N,N-trimethyl-ammonium bromide (CTAB) use amphipatic ammonium salts which contain a longer hydrocarbon chain of at least 6 C atoms.
B) Lysis with water: During the lysis with water, the properties of semipermeable membranes of cells are used which envelope these. These membranes are water-permeable, but not for salts or larger molecules. If a cell with its contents of salt and other larger molecules is transferred into a fluid, which contains a lower salt concentration than the cell interior, the cell will absorb water until the salt concentration within and outside the cell is balanced. If the concentration difference between the cell interior and the outer fluid is sufficiently large, the cell will absorb water until it bursts.
C) Lysis with organic solvents: the hydrophobic membranes of cells can be destroyed by organic solvent. The lipids of the membranes and the lipophilic protein components of the membranes are hereby absorbed in the organic phase. The cell contents remain mostly in the hydrophilic phase or at the boundary layer between the lipophilic and the hydrophilic phase. Phenol is often used with this type of lysis.
D) Lysis with chaotropic salts: chaotropic salts destroy the structure of water based on the formation of hydrogen bridge bonds, so that the double layer structure of membranes cannot be maintained anymore. These membranes are thereby dissolved and the lysis takes place, whereby the cell contents pour into the chaotropic environment. This method is used with a number of methods for isolating nucleic acids and proteins, e.g. in the commercially available RNeasy®-, QIAamp®-methods or the denaturating disintegration of cells for protein cleaning.
It is desirable for the following cleaning steps or detection reactions which are carried out with the obtained lysate that the relevant contents of the cell are transferred as completely as possible into the lysate, as the amount which is necessary for the detection or for the isolation of a given amount of a biomolecule such as DNA or RNA, can be reduced further.
With the quantitative and the qualitative analysis of biomolecules as e.g. nucleic acids and proteins, the preservation of their integrity is of great importance. In particular nucleic acid such as DNA and, to a higher extent, RNA are subject to different influences after the removal of the biological samples from their natural environment, which can lead to a change or degradation of the DNA or RNA. For example, the enzymatic degradation of these nucleic acids or the degradation of DNA under the influence of shear forces occurring in the sample during the lysis are mentioned.
It is known that the preservation of the integrity of nucleic acids and proteins can be achieved by (A) cleaning of the NA or proteins, (B) dehydration, (C) protection against degrading enzymes or (D) complexing. This is described shortly in the following:
Cleaning of the nucleic acids or proteins: during the cleaning, nucleic acids or proteins are freed from all substances or molecules which are contained in the biological sample and which can damage the integrity of the nucleic acids or the proteins permanently. These cleaning methods are e.g. affinity chromatography, protein salting out methods, cleaning of nucleic acids or proteins at solid phases. The solid phase can e.g. be an ion exchanger as is described in U.S. Pat. Nos. 5,990,301, and 6,020,186 for example. Alternatively, the use of porous matrices is described, e.g. in U.S. Pat. No. 6,180,778 or U.S. Pat. No. 5,496,562.
(B) Dehydration: a dehydration of the nucleic acid or proteins causes that damaging processes, which can take place when the sample with the nucleic acids or proteins are dissolved in water, are blocked or prevented. These damaging processes are e.g. the enzymatic degradation by proteases or nucleases. Thus, the isolated nucleic acid can be dehydrated e.g. by precipitation (precipitation) by means of salt and alcohol (Current protocols in molecular biology, e.g. p. 1.6.1. Alkaline Lysis Miniprep). Nucleic acids can also be protected from degradation in biological samples as e.g. in tissue parts or microscopic sections by dehydration. This is described in the description U.S. Pat. No. 6,528,641 for example. The reagents are infiltrated into the intact sample in the method described there, whereby not only a dehydration, but also a precipitation of proteins as e.g. nucleases is effected, which are then present in the dehydrated sample in an inactive manner. The use of ammonium sulfate as dehydrating agent is particularly described there. A precipitation of DNA is also effected in the cleaning of DNA described in the patent application US 2002/0197637, whereby polyamines are used for cleaning and the polyamines lead to a condensation of the DNA, whereby a damage of the DNA by shearing shall be avoided with mechanical disintegration.
(C) Protection from degrading enzymes: it has been known for a long time that proteases and nucleases (enzymes disintegrating nucleic acid) can be specifically inhibited. Thus, the DNase I can be inhibited by e.g. Mg2+- or Ca2+-complexing agents. RNases can be inhibited e.g. by specific RNase inhibitors or by reducing agents. These inhibitors are described in the U.S. Pat. Nos. 5,552,302 and 6,777,210.
(D) Complexing: Another variant of the cleaning provides a complexation with quaternay ammonium salts forming micelles for the protection of the nucleic acids. The complexing of the nucleic acids thus leads to the protection from nucleases. Quaternary ammonium salts are used thereby, which function as cationic detergents and form micelles. For this it is necessary that the quaternary ammonium salt has at least one long-chain carbon chain as substituent. The U.S. Pat. Nos. 6,602,718, 6,617,170, 5,010,183, 5,985,572, 5,300,635, 5,728,822 and the US patent applications US 2002/0146677A1, US 2004/0048384A1, US 2004/0115689, and US 2004/0014703A1 are counted amongst these or similar methods.
U.S. Pat. No. 6,821,752 describes a cleaning and extraction of proteins in the presence of amphipatic amines, that is, of compounds acting as detergents in a similar manner.
The compounds provided for the separation or complexation of the nucleic acids or proteins is restricted in that they should behave as inert as possible with regard to the isolation method or analysis method carried out subsequently, that is, should not influence the subsequent isolation or analysis in a disadvantageous manner. Otherwise, they have to be decomplexed in a further reconditioning step.
The above state of the art shows that sufficient stabilisation of nucleic acids and proteins from biological samples usually requires additional reconditioning steps by which the sample is split. Methods for the stabilisation which make these additional reconditioning steps unnecessary and which use stabilising reagents which can be used as versatile as possible, are therefore advantageous.
A further problem which can occur with the analysis of biomolecules such as nucleic acids or samples containing proteins, is the fact that biomolecules of a first type can be impaired by biomolecules of a second type in such a sample. Sometimes, biomolecules of the second types shall also be analysed, whereby biomolecules of the first or another type will then act in a disrupting manner during the analysis. An example amongst many is mentioned here to clarify this: A sample contains cells of a certain organism. A detection of a nucleic acid of these cells by an oligonucleotide in the presence of proteins binding nucleic acid can for example be influenced in a disadvantageous manner. Assuming that a certain species of DNA is to be detected in the present example, the proteins binding the DNA represent an inhibiting substance. In another example, e.g. the analysis of proteins binding DNA, a DNA which can bind to the proteins binding DNA can have a disadvantageous influence on the result of the analysis, as the nucleic acid acts as inhibiting substance in this case and leads to a falsification of the results of the analysis.
Usual methods for excluding these disadvantageous interactions between different biomolecules provide to separate the biomolecules which are influencing each other to avoid disturbances. These methods thus often provide a cleaning step, in which certain species of biomolecules are removed from a sample. The concentration of the biomolecules amongst themselves is thereby changed by the separation method, so that the desired biomolecules are enriched, but the other ones are reduced in their concentrations.
Known methods for the separation of biomolecules include the following methods:
Separation of biomolecules by salting out methods: Salting out methods are used to separate parts of the biological sample which can be precipitated by a certain amount of salts from the sample. These methods can be used on the one hand to precipitate the part to be cleaned with salt. The ammonium sulfate precipitation of proteins represents such a known method. On the other hand, this method can also be used in the reverse direction, an that the material to be cleaned is freed from a plurality of contaminating molecules, but is not precipitated itself. An example for this is the protein precipitation by potassium acetate during the isolation of DNA.
Separation of biomolecules by chromatography: Biomolecules can, due to their properties, be cleaned and separated by several chromatography methods. These properties can concern their size, their charge, their hydrophobity or their affinity to certain surfaces or haptenes, to mention only a few possibilities. Chromatography is to be explained here with the example of the ion exchange chromatography: There are two possibilities for a series of chemical biomolecules to be cleaned by ion exchange chromatography. The biomolecule to be isolated can on the one hand be bound to the ion exchange chromatography material, whereas the contaminating substances are not bound and can be separated from the biomolecule to be isolated in this manner (e.g. anion exchange chromatography for the cleaning of negatively charged nucleic acids). On the other hand, contaminating substances can also be bound to the chromatography material, and the biomolecule to be isolated can then be caught in the break-through or in the washing buffer (e.g. cation exchange chromatography for the cleaning of the negatively charged nucleic acids). Examples of such a method are described in U.S. Pat. No. 5,990,301 and WO0248164.
(C) Separation of biomolecules at solid surfaces: A number of surfaces have the property to be able to bind certain biomolecules in a defined binding environment. Theses binding properties can be used for the cleaning of biomolecules. This will be explained here with the example of the cleaning of nucleic acid at silica surfaces. Silica surfaces, be it microparticles or membranes, can bind nucleic acids in the presence of chaotropic salts having a high concentration. But other molecules as e.g. proteins do not bind to these surfaces and are thus separated. The nucleic acid can be obtained in a pure form after the washing of the silica surface. Examples for this are described in the U.S. Pat. Nos. 5,990,301, 6,020,186 and 6,180,778.
(D) Separation of biomolecules by selective complexing: Some separation methods complex biomolecules selectively, so that these can be separated from the other contents of the sample by a centrifugation step or by a filtration step. Examples for this are described in U.S. Pat. Nos. 5,728,822 and 5,985,572.
The use of ammonium sulfate for the neutralisation of inhibiting effects at samples containing RNA is described in the US patent application US 2002/0115851. These samples contain purified amounts of RNA. However, it is described in the literature that inorganic ammonium salts as e.g. ammonium sulfate have the disadvantage that certain partial activities of polymerases (e.g. 3′-5′ exonuclease activity) can be changed and the presence of ammonium sulfate can thereby have a disadvantageous effect on the polymerase reactions carried out for the subsequent analysis (Tsurumi et al. “Functional Expression and Characterization of the Epstein-Barr Virus DNA Polymerase Catalytic Subunit”, Journal of Virology, Vol 67, No. 8, 1993, p. 4651-4658)
The described methods require a preceding separation of the biomolecules affecting the detection reaction in a disadvantageous manner from the sample or presume these. Consequently, a need exists for a simplified method with which the inhibiting effect of certain biomolecules in samples can be removed in such a measure that a reliable detection reaction can be carried out with the sample.
A further problem which often occurs during the separation of nucleic acids, is founded in the fact that ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) represent biomolecules which are very similar in their chemical properties. Thus, there is often the difficulty to separate these from each other. For a plurality of applications, it is nevertheless of utmost importance to produce DNA-free RNA or RNA-free DNA: contaminating genomic DNA in RNA preparations can for example lead to quantitatively wrong results in RT-PCR experiments.
At present, a number of methods exist which make it possible to enrich RNA or DNA differentially. Different methods can be distinguished in principal:
On the one hand, enzymes can be used which specifically degrade DNA or RNA. These enzymes are called nucleases. The RNA-degrading enzymes belong to these nucleases, the RNases, and the DNA-degrading enzymes, the DNases. If e.g. DNA is to be cleaned, a RNase can be used during the cleaning method, which decomposes the RNA molecules into small fractions. These methods are generally used for e.g. plasmid isolation or cleaning of genomic DNA. DNA contaminations in RNA preparations are alternatively hydrolysed with DNase I. This method is also generally known.
On the other hand, chemical methods can be used which utilise the differences in the chemical properties of RNA or DNA. A number of solid phase cleaning methods are counted amongst these. The solid phase can e.g. be an ion exchanger, as for example described in U.S. Pat. Nos. 5,990,301 and 6,020,186, or it can be a porous matrix, as is described in e.g. U.S. Pat. Nos. 6,180,778 and 5,496,562.
Other cleaning methods concern the different solubility behaviours of RNA or DNA. The cleaning of RNA in the presence of aqueous solution of a chaotropic salt and acidic phenol is counted amongst these, whereby genomic DNA enriches in the inter phase and RNA remains in aqueous solution (Chomczynski P. & Sacchi N., 1987, Anal Biochem. 1987 April; 162(1):156-9, “Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction”).
Other methods are based on the selective precipitation of RNA or DNA. Examples for this have already been described above in connection with the decomplexing of inhibiting biomolecules or the stabilisation of biomolecules.
These methods also include a separate method step, with which the relative composition of the different biomolecules, in particular different nucleic acids or proteins is changed considerably to achieve the desired effect. The practicability of the method can depend if the reagant used for the separation has a negative effect on the desired detection reaction, whereby the number of reagents available for a certain sample preparation as e.g. for an analysis method is restricted. Every method step for the separation of a sample also has a contamination risk in addition to the additional costs, which makes an extremely clean and controlled operation necessary.
With regard to different aspects, there is thus a need for a further improvement of the sample preparation of samples containing biomolecules and improved processing, preparation, and analysis methods of biomolecules.